JP7088511B2 - Composite molding composition containing fibroin-like protein and method for producing the same - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law 1. Publisher name: ACS Publications Publication name: Biomacromolecules 2017 (pages 1002 to 1009) Date of publication: January 24, 2017 (2017) https: // pubs. acs. org / doi / 10.1102 / acs. biomac. 6b01891

The present invention relates to a composite molding composition containing a fibroin-like protein and a method for producing the same.

Natural spider silk made of fibroin has excellent properties against physical changes such as tensile strength, toughness and extensibility, for example, toughness compared to iron of the same weight. It is a polypeptide fiber having about 5 times. In addition, it has excellent heat resistance, and since it is formed of a polypeptide, it has high biocompatibility and biodegradability. However, unlike silk moths that use natural threads industrially, spiders are difficult to breed in large quantities, so we manufacture artificial synthetic fibers that imitate spider threads, and have the characteristics against physical changes and biocompatibility. Attempts have been made to apply it to materials such as threads and mattresses having properties and biodegradability (Patent Documents 1 to 5). However, the production of artificial fibers comparable to natural spider silk in terms of the above properties has not been successful.

Natural spider silk has a core portion in which polyalanine and glycine-rich polar regions that form a β-sheet structure are repeated in its structure, and highly conserved non-repetitive amino ends and carboxyl on both sides thereof. Due to the arrangement of the terminal domains, it has a random coil-like structure, a β-sheet structure and a helix structure, and the mixture of these structures is considered to be the reason for the excellent properties of natural spider silk, and its structural properties are considered to be the reason. It has been studied (Non-Patent Documents 1 to 3). Also, when spun in the body of a spider, unlike normal proteins, spider proteins are highly soluble when stored at high concentrations, but change to extremely durable fibers as needed. (Non-Patent Document 2). However, the structural characteristics and the spider silk production mechanism have not always been clarified.

The present inventor clarified the manufacturing process of the traction thread in the traction thread gland of the spider spider, and clarified that the formation of the granular structure by the β-sheet structure of the spider thread fibroin protein is important for exerting the physical properties of the spider thread. (Patent Document 6).

International Publication WO 2007/078239 International Publication WO2010 / 123450 International Publication WO 2011/112046 International Publication WO2012 / 165476 International Publication WO 2013/065650 International Publication WO2017 / 030197

Numata K et al., Soft Matter June 30, 2015, 11, 6335-6342 Hagn F et al., Nature. 2010; 465 (7295): 239-242 Teule F et al., Nature Protocols 2009; 4: 341-355

Based on the production mechanism of natural spider traction threads in the spider body and the factors that bring about high properties, by applying this mechanism to the production of artificial fibers, it contains fibroin-like proteins with properties such as high strength and toughness. Improve the physical properties of the composite molding composition, the method for producing the composition, the composite molding composition such as the composite fiber, the composite film and the composite resin produced by the production method, and the composite molding composition. Provide a method.

The present inventors have prepared a composite molding composition produced by blending a natural fibroin protein or an artificially produced fibroin-like protein with a polypeptide or polyamino acid having a β-sheet structure and molding the β-sheet structure. The present invention has been completed by finding that it exhibits excellent physical properties such as tensile strength, strain and toughness as compared with a molding composition containing no polypeptide or polyamino acid having a sheet structure.

Specifically, the present invention provides a composition containing a fibroin-derived protein and a polypeptide having a β-sheet structure and / or a polyamino acid.

Further, the composition may be a composite molding composition in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein and molded.

The composite molding composition may be a composite molding composition in which a β-sheet structure derived from the polypeptide and / or a polyamino acid is retained.

The composite molding composition may be a composite molding composition selected from a composite fiber, a composite film, a composite gel, or a composite molded body.

Further, the present invention is a composite molding composition in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein.
The composite molding composition is selected from composite fibers, composite films or composite gels.
(i) A step of preparing a dope solution in which a fibroin-derived protein and a polypeptide having a β-sheet structure and / or a polyamino acid are dissolved.
(ii) A step of molding a composite molding composition from the dope solution,
Provided is a composite molding composition produced by a production method including.

Further, the present invention is a composite molding composition, wherein the composite molding composition is selected from composite fibers or composite films.
In addition to steps (i) and (ii) above,
(iii) The step of stretching the composite molding composition obtained in (ii) above in a solvent and drying it.
Provided is a stretched composite molding composition produced by a production method comprising the above.

Further, the present invention is a composite molding composition, and the composite molding composition is a molded body.
(i) A step of mixing a fibroin-derived protein with a polypeptide having a β-sheet structure and / or a polyamino acid to prepare a mixture.
(ii) The step of applying pressure to the mixture and heating it,
Provided is a composite molding composition which is a composite molded body manufactured by a manufacturing method including.

In the composite molding composition of the present invention, the fibroin-derived protein is used.
(i) Natural fibroin-derived protein and / or
(ii) May be selected from modified fibroin-derived proteins.

In the composite molding composition of the present invention, the natural fibroin-derived protein is derived from silk fibroin-derived silk protein (silk fibroin protein), spider silk fibroin-derived spider silk protein (spider silk fibroin protein), and hornet silk fibroin. It may be at least one selected from the group consisting of hornet silk proteins.

ただし、前記ドメイン配列が、天然由来のフィブロインと比較して、少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されており、グリシン残基の含有量が低減されたアミノ酸配列を有する、改変フィブロインタンパク質：
[式I中、
（Ａ）_ｎモチーフは２～２０アミノ酸残基から構成されるアミノ酸配列を示し、かつ、
（Ａ）_ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が４０％以上であり、
ＲＥＰは２～２００アミノ酸残基から構成されるアミノ酸配列を示し、
ｍは２～３００の整数であり、
複数存在する（Ａ）_ｎモチーフは、相互に同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよく、
複数存在するＲＥＰは、相互に同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい]
コンポジット成形組成物である場合がある。In the composite molding composition of the present invention, the modified fibroin-derived protein is a modified fibroin-derived protein containing a domain sequence represented by the following formula (I);

However, in the domain sequence, at least one or more glycine residues in REP are replaced with different amino acid residues as compared with naturally occurring fibroin, and the content of glycine residues is reduced. Modified fibroin protein with sequence:
[In Formula I,
(A) _{The n} motif indicates an amino acid sequence composed of 2 to 20 amino acid residues, and
(A) The number of alanine residues is 40% or more of the total number of amino acid residues in the _n motif.
REP shows an amino acid sequence consisting of 2 to 200 amino acid residues.
m is an integer from 2 to 300
Multiple (A) _n motifs may have the same amino acid sequence or different amino acid sequences.
Multiple REPs may have the same amino acid sequence or different amino acid sequences.]
It may be a composite molding composition.

In the modified fibroin-derived protein of the composite molding composition of the present invention, the domain sequence represents GGX and GPGXX in REP (where X represents an amino acid residue other than glycine) as compared to naturally occurring fibroin. In at least one motif sequence selected from.), It has an amino acid sequence corresponding to one or more glycine residues in the motif sequence being replaced with another amino acid residue, and has a glycine residue. When the ratio of the motif sequence in which the group is replaced with another amino acid residue is 10% or more, preferably 20% or more, more preferably 30% or more, and most preferably 40% or more with respect to the total motif sequence. There is

In the modified fibroin-derived protein, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared from the N-terminal side to the C-terminal side, and the amino acid residues are compared. When the number of amino acid residues of the REP having a small number is 1, the ratio of the number of amino acid residues of the other REP is 2 to 3.5. The amino acid residue of the two adjacent [(A) _n motif-REP] units. When the total value obtained by adding the radix is x and the total number of amino acid residues in the domain sequence is y, the maximum value of x / y (%) may be 20% or more.

In the composite molding composition, the modified fibroin-derived protein represented by the above formula (I) may be a modified fibroin-derived protein having an amino acid sequence represented by SEQ ID NOs: 1 to 19.

In the composite molding composition of the present invention, the fibroin-derived protein has 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more homology with respect to the amino acid sequence of the fibroin-derived protein. It may be a fibroin-derived protein having sex.

In the composite molding composition of the present invention, the polyamino acid having the β-sheet structure may be polyalanine.

In the composite molding composition of the present invention, the polyalanine may be selected from liner-type polyalanine (L-polyAla) or telechelic-type polyalanine (T-polyAla).

Further, the present invention is a method for producing a composite molding composition selected from a composite fiber, a composite film, and a composite gel, in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein.
(i) A step of preparing a dope solution in which a fibroin-derived protein and a polypeptide having a β-sheet structure and / or a polyamino acid are dissolved.
(ii) A step of producing the composite molding composition by spinning or stretching the dope solution.
To provide a manufacturing method including.

Further, the present invention is a method for producing a composite molding composition of a molded product, in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein.
(i) A step of mixing a powdery fibroin-derived protein with a polypeptide having a powdery β-sheet structure and / or a polyamino acid to prepare a mixture.
(ii) The step of applying pressure to the mixture and heating it,
To provide a manufacturing method including.

Further, the present invention is a method for producing a stretched composite molded composition.
(i) A step of stretching the composite molding composition produced by the above-mentioned production method in a solvent.
as well as,
(ii) A step of drying the stretched composite molded composition,
To provide a manufacturing method including.

In the production method of the present invention, the fibroin-derived protein is
(i) Natural fibroin-derived protein and / or
(ii) May be selected from modified fibroin-derived proteins.

In the production method of the present invention, the natural fibroin-derived protein is silk fibroin-derived silk protein (silk fibroin protein), spider silk fibroin-derived spider silk protein (spider silk fibroin protein), and hornet silk fibroin-derived hornet silk. It may be at least one selected from the group consisting of proteins.

ただし、前記ドメイン配列が、天然由来のフィブロインと比較して、少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されており、グリシン残基の含有量が低減されたアミノ酸配列を有する、改変フィブロインタンパク質：
[式I中、
（Ａ）_ｎモチーフは２～２０アミノ酸残基から構成されるアミノ酸配列を示し、かつ、
（Ａ）_ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数が４０％以上であり、
ＲＥＰは２～２００アミノ酸残基から構成されるアミノ酸配列を示し、
ｍは２～３００の整数であり、
複数存在する（Ａ）_ｎモチーフは、相互に同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよく、
複数存在するＲＥＰは、相互に同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい]。In the production method of the present invention, the modified fibroin-derived protein may be a modified fibroin-derived protein containing a domain sequence represented by the following formula (I);

However, in the domain sequence, at least one or more glycine residues in REP are replaced with different amino acid residues as compared with naturally occurring fibroin, and the content of glycine residues is reduced. Modified fibroin protein with sequence:
[In Formula I,
(A) _{The n} motif indicates an amino acid sequence composed of 2 to 20 amino acid residues, and
(A) The number of alanine residues is 40% or more of the total number of amino acid residues in the _n motif.
REP shows an amino acid sequence consisting of 2 to 200 amino acid residues.
m is an integer from 2 to 300
Multiple (A) _n motifs may have the same amino acid sequence or different amino acid sequences.
A plurality of REPs may have the same amino acid sequence or different amino acid sequences.]

In the modified fibroin-derived protein of the production method of the present invention, the domain sequence is GGX and GPGXX in REP as compared with naturally occurring fibroin (where X represents an amino acid residue other than glycine). In at least one motif sequence selected from, the glycine residue has an amino acid sequence corresponding to the substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue, and the glycine residue is The proportion of the motif sequence substituted with another amino acid residue may be 10% or more, 20% or more, more preferably 30% or more, and most preferably 40% or more with respect to the total motif sequence.

In the modified fibroin-derived protein of the production method of the present invention, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared from the N-terminal side to the C-terminal side. Then, when the number of amino acid residues of REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues of the other _REP is 2 to 3.5. When the maximum value of x / y (%) is 20% or more when the total value obtained by adding the number of amino acid residues of the REP] unit is x and the total number of amino acid residues of the domain sequence is y. There is.

In the production method of the present invention, the modified fibroin-derived protein represented by the above formula (I) may be a modified fibroin-derived protein having an amino acid sequence represented by SEQ ID NOs: 1 to 19.

The fibroin-derived protein has 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more homology with respect to the amino acid sequence of the fibroin-derived protein represented by SEQ ID NOs: 1 to 19. It may be a fibroin-derived protein having.

In the production method of the present invention, the polyamino acid having the β-sheet structure may be polyalanine.

In the production method of the present invention, the polyalanine may be selected from liner-type polyalanine or telechelic-type polyalanine.

Further, the present invention provides a method for improving the physical properties of a composite molding composition by blending a polypeptide having a β-sheet structure and / or a polyamino acid with a fibroin-derived protein.

The present invention further improves the physical properties of the composite molding composition by further improving the physical properties of the composite molding composition, and further, by stretching the composite molding composition in a solvent and then drying it. Provide a way to make it.

In the method of improving the physical properties of the composite composition, the improvement of the physical properties may be an increase in tensile strength, an increase in strain and / or an increase in toughness.

Provided are composite fibers containing fibroin-like polypeptide fibers having physical properties such as high toughness, strain (displacement at break point) and tensile strength (stress), composite molding compositions such as composite films and composite gels, and methods for producing the same. can.

It is a schematic diagram which shows the domain sequence of modified fibroin. It is a figure which shows the distribution of the value of x / y (%) of the fibroin of natural origin. It is a figure which shows the distribution of the value of z / w (%) of the fibroin of natural origin. It is a figure which shows the outline of the spinning method from the dope liquid for spinning the composite fiber which is the composite molding composition of this invention. Changes in tensile strength, strain and toughness of a stretched composite film produced by blending L-polyAla or T-polyAla in various proportions with natural silk moth fibroin protein and stretching it at a stretch rate of 100%. It is a figure which shows the result of having measured. A stretched composite film produced by blending T-polyAla at a ratio of 5 wt% with ADF3 Kai-noNR, which is a modified spider silk fibroin protein, and stretched in methanol at various stretching ratios, is used for tensile strength. It is a figure which shows the result of having measured the change of strength, strain and toughness. Wide-angle X-ray scattering (WAXD) for a composite film produced by blending L-polyAla or T-polyAla in various ratios with ADF3 Kai-noNR, which is a modified spider silk fibroin protein, and stretching it at a stretching rate of 100%. It is a figure which shows the result of having measured.

1. 1. Composite molding composition 1-1. Non-stretched composite molding composition One of the embodiments of the present invention is a composition containing a fibroin-derived protein and a polypeptide having a β-sheet structure and / or a polyamino acid, and in particular, the composition is composite molding. It is a non-stretched composite molding composition which is a composition in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein and molded.

Using this non-stretched composite molding composition, the stretched composite molding composition described later can be produced.

As an example of the composite molding composition, a β-sheet structure derived from the polypeptide and / or a polyamino acid is retained, and the composite molding composition is made from a composite fiber, a composite film, a composite gel or a composite molded body. Examples include the composite molding composition of choice.

(1) Fibroin-derived protein Fibroin is known as a general term for fibrous proteins that constitute silk such as insects and spiders and occupy 70% of the silk. As used herein, "fibroin" also includes natural silks of insects and spiders, and artificially produced silks that mimic or direct the composition and properties of these natural silks. In the present specification, "fibroin" includes the following spiderin proteins such as spiderin I and II in "fibroin", but not only fibroin derived from these spider silks but also fibroin derived from silk moth silk yarn and the like. included. In addition to natural fibroin, fibroin produces, for example, a nucleic acid sequence encoding a desired amino acid sequence by a gene recombination method, incorporates this into an expression vector, and prepares a recombinant expression vector by a known method. Transformants can be prepared by introducing them into suitable hosts such as bacteria, yeast, mammalian cells, plants, and insect cells, and these can be isolated, purified, and used (International Publication WO2012 / 165476, etc.). Also, some of the fibroin is commercially available, for example, they can be obtained and used.

The fibroin-derived protein according to the present invention is not particularly limited, and may be a natural fibroin, or a so-called natural fibroin-derived protein produced by a microorganism or the like by a gene recombination technique or synthetically produced. It may be a protein.

Further, such fibroin-derived protein comprises, for example, silk fibroin-derived silk protein (silk fibroin protein), spider silk fibroin-derived spider silk protein (spider silk fibroin protein), and hornet silk fibroin-derived hornet silk protein. It may be one or more selected from the group. Among these, spider silk protein derived from spider silk fibroin is preferably used.

As used herein, the term "fibroin-derived protein" refers to a protein constituting naturally-derived fibroin such as the above-mentioned insects and spiders, and other than these naturally-derived proteins, for example, artificially produced by a gene recombination method. Contains proteins that are similar in structure and properties to the fibroin produced.

A protein having a similar structure to fibroin means that the amino acid sequence of natural fibroin is 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more. , And protein fragments having a partial sequence of these proteins, or fusion proteins of these proteins or protein fragments with other proteins or peptides. Further, in the present specification, "fibroin-derived protein" may be referred to as "fibroin-derived polypeptide" based on its structural properties.

Further, a protein having similar properties to fibroin has at least one property selected from the tensile strength, toughness and elasticity of natural fibroin, which is at least 80%, preferably 85% or more, as compared with natural fibroin. , More preferably 95% or more, most preferably 100% or more. Further, in the present specification, when describing the structure and physical properties of such fibroin, it may be described as "fibroin-like polypeptide" or "fibroin-like protein".

As the fibroin, the following modified fibroin can also be used. The modified fibroin is a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids. The protein containing the domain sequence represented by this formula (I): [(A) _n motif-REP] _m is described in detail in PCT / JP2017 / 016917 and PCT / JP2017 / 016925, and is described herein by reference. Is taken in by.

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of the naturally occurring fibroin, or may be a fibroin having the same amino acid sequence as the naturally occurring fibroin. As used herein, "naturally occurring fibroin" is also a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m .

The "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as it is, as long as it has the amino acid sequence specified in the present invention, and relies on the amino acid sequence of naturally occurring fibroin. The amino acid sequence may be modified (for example, the amino acid sequence may be modified by modifying the gene sequence of cloned naturally occurring fibroin), and artificially designed and artificially designed regardless of naturally occurring fibroin. It may be synthesized (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically to the REP of the amino acid sequence). It is an amino acid sequence that produces (corresponding to)), and means an amino acid sequence represented by the formula (I): [(A) _n motif-REP] _m . Here, (A) the _n motif indicates an amino acid sequence mainly composed of an alanine residue, where n is 2 to 20, preferably 4 to 20, more preferably 8 to 20, still more preferably 10 to 20, and even more. It may be an integer of preferably 4 to 16, even more preferably 8 to 16, and particularly preferably 10 to 16. Further, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, preferably 60% or more, more preferably 70% or more, and 80%. It is more preferably% or more, still more preferably 90% or more, and may be 100% (meaning that it is composed only of alanine residues). REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. m represents an integer of 2 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences. Specific examples of the protein derived from the large spit tube bookmark thread include proteins containing the amino acid sequences represented by SEQ ID NOs: 1 to 3 which are partial sequences of the natural sequence and SEQ ID NOs: 4 to 19 which are modified natural sequences. Can be done.

The amino acid sequence (Met-PRT313) shown in SEQ ID NO: 6 is a region "continuing alanine residues" in the amino acid sequence of the naturally occurring fibroin Nephila clavipes (GenBank accession number: P468401, GI: 11744415). It has a sequence in which amino acid residues have been modified, such as deleting the number of consecutive alanine residues in "(A) _n motif" to five. The amino acid sequence shown by SEQ ID NO: 12 (PRT313) is obtained by adding the amino acid sequence (tag sequence and hinge sequence) shown by SEQ ID NO: 21 to the N-terminal of the amino acid sequence shown by SEQ ID NO: 6 (Met-PRT313). be.

The amino acid sequence (Met-PRT399) shown in SEQ ID NO: 7 is obtained from the amino acid sequence shown in SEQ ID NO: 6 every other (A) _n motif ((A) ₅ ) from the N-terminal side to the C-terminal side. Is deleted, and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence, and the amino acid sequence (PRT399) shown in SEQ ID NO: 13 is the amino acid represented by this SEQ ID NO: 7. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 21 is added to the N-terminal of the sequence.

The amino acid sequence (Met-PRT380) shown in SEQ ID NO: 8 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 14 (PRT380). Is obtained by adding the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 21 to the N-terminal of the amino acid sequence shown in SEQ ID NO: 8.

The amino acid sequence (Met-PRT410) shown in SEQ ID NO: 9 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 7, and the amino acid sequence shown in SEQ ID NO: 15 (PRT410). Is obtained by adding the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 21 to the N-terminal of the amino acid sequence shown in SEQ ID NO: 9.

In the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT468), two residues of A are inserted into the poly A region in SEQ ID NO: 6 with respect to the amino acid sequence shown by SEQ ID NO: 9, and the amino acid shown by SEQ ID NO: 9 is used. The amino acid sequence (PRT468) shown in SEQ ID NO: 16 is the amino acid sequence (PRT468) represented by SEQ ID NO: 16, in which two repeated sequences on the C-terminal side were deleted and replaced with S or P at 13 locations so that the molecular weight of the sequence was almost the same. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 21 is added to the N-terminal of the amino acid sequence represented by No. 10.

The amino acid sequence shown in SEQ ID NO: 11 (Met-PRT799) contains several amino acid residues on the C-terminal side of the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 9 are repeated four times. , The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 21 is added, and the amino acid sequence (PRT799) shown in SEQ ID NO: 17 is the N-terminal of the amino acid sequence shown in SEQ ID NO: 11. The amino acid sequence (tag sequence and hinge sequence) shown in 21 is added.

The above amino acid sequences are summarized below.
SEQ ID NO: 1 Amino acid sequence of Araneus diadematus SEQ ID NO: 2 Amino acid sequence of Araneus diadematus SEQ ID NO: 3 Amino acid sequence of Araneus diadematus SEQ ID NO: 4 Amino acid sequence of recombinant spider silk protein ADF3 KaiLargeNRSH1 SEQ ID NO: 5 The amino acid sequence of His tag was deleted from SEQ ID NO: 4. SEQ ID NO: 6 Amino acid sequence of Met-PRT313: Met-CRY1_L_A5 Amino acid sequence of SEQ ID NO: 7 Met-PRT399: Met-CRY1_L_A5_giza Amino acid sequence of SEQ ID NO: 8 Met-PRT380: Met-CRY1_L_A5_QQQ Amino acid sequence SEQ ID NO: 9 Met-PRT410 Amino acid sequence of SEQ ID NO: 10 Met-PRT468: Met-CRY1_L_A7_giza_QQQ amino acid sequence SEQ ID NO: 11 Met-PRT799: Met-CRY1_200_A5_giza_QQQ_WHis6 amino acid sequence SEQ ID NO: 12 PRT313 amino acid sequence SEQ ID NO: 13 PRT399 amino acid sequence SEQ ID NO: 15 Amino acid sequence of PRT410 Amino acid sequence of SEQ ID NO: 16 PRT468 Amino acid sequence of SEQ ID NO: 17 PRT799 Amino acid sequence of SEQ ID NO: 18 ADF3Kai_noNR Amino acid sequence of SEQ ID NO: 19 Amino acid sequence of SEQ ID NO: 18 excluding His tag SEQ ID NO: 20 His tag And amino acid sequence of start codon Amino acid sequence of SEQ ID NO: 21 His tag

The modified fibroin is, for example, modifying the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. Can be obtained at. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by a method well known to those skilled in the art such as a partially specific mutagenesis method. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Enzymeology, 100, 448 (1983).

The naturally occurring fibroin is a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m , and specific examples thereof include fibroin produced by insects or spiders. Be done.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama) Silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

Examples of the fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Torinofundamashi (genus Cyrtarachne), spiders belonging to the genus Trinofundamashi, and spiders belonging to the genus Cyrtarachne. Spiders belonging to (Gasteracantha genus), spiders belonging to the spider genus Ordgarius such as Mameitaiseki spider and Mutsutogei sekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, and spiders belonging to the genus Cytophora. ) Spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Tetragnatha, such as the spider Yasagata spider, Harabiroashidakagumo, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha such as spiders, spiders such as spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the family Spiders (Tetragnathidae) such as spiders belonging to the genus Euprostenops. Examples include pider silk protein (spider silk protein). Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2) and the like.

More specific examples of fibroin produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-. 4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence Amino acid 04) U37520 (base sequence)), major amplify protein 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein2 (Amino acid sequence), AF441245 (base sequence)), major amplifier spidroin 1 [derived from Europe protein australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major amplifier Spinbolte protein 2 Accession number CAM3224.91 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amplifier silk protein (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR) 3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally derived fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidoline, amplify, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The modified fibroin may be modified silk fibroin (modified amino acid sequence of silk protein produced by spiders) or modified spider silk fibroin (modified amino acid sequence of spider silk protein produced by spiders). There may be. Among them, modified spider silk fibroin is preferably used.

As a specific example of the modified fibroin, the content of the modified fibroin [modified fibroin according to the first embodiment] derived from the large spider tube bookmark thread protein produced by the spider's large bottle-shaped line and the glycine residue is reduced. Modified fibroin [modified fibroin according to the second embodiment], (A) modified fibroin with reduced content of _n motif [modified fibroin according to the third embodiment], content of glycine residue, and (A) Examples thereof include modified fibroin [modified fibroin according to the fourth embodiment] in which the content of _n motifs is reduced.

Examples of the modified fibroin according to the first embodiment include proteins containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . In the modified fibroin according to the first embodiment, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, and an integer of 10 to 20 in the formula (I). Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the modified fibroin according to the first embodiment, the number of amino acid residues constituting REP in the formula (I) is preferably 10 to 200 residues, more preferably 10 to 150 residues. , 20-100 residues are even more preferred, and 20-75 residues are even more preferred. The modified fibroin according to the first embodiment is a total residue of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula (I): [(A) _n motif-REP] _m . The number of groups is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number of amino acid residues.

The modified fibroin according to the first embodiment contains a unit of an amino acid sequence represented by the formula (I): [(A) _n motif-REP] _m , and the C-terminal sequence is any one of SEQ ID NOs: 1 to 3. Amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more homology with the amino acid sequence shown in 1 or any of SEQ ID NOs: 1 to 3. It may be a polypeptide that is.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C end of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C end of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the modified fibroin according to the first embodiment, (1-i) the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 (amino acid sequence obtained by removing the His tag from the amino acid sequence of SEQ ID NO: 4). , Or (1-ii) contains an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. Modified fibroin can be mentioned. The sequence identity may be 100%.

The amino acid sequence represented by SEQ ID NO: 4 is the first to the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 20) consisting of a starting codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 8.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

In the modified fibroin according to the second embodiment, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more with respect to the total motif sequence.

The modified fibroin according to the second embodiment contains a domain sequence represented by the formula (I): [(A) _n motif-REP] _m , and is located most C-terminal to the above domain sequence (A). The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence from the _n motif to the C-terminal of the above domain sequence. Let z be the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side of the domain sequence to the C-terminal of the domain sequence. It may have an amino acid sequence in which w (%) is 30% or more, or may have an amino acid sequence in which w (%) is 50.9% or more. (A) The number of alanine residues with respect to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

It is preferable that the modified fibroin according to the second embodiment has an increased content ratio of the amino acid sequence consisting of XGX by substituting one glycine residue of the GGX motif with another amino acid residue. In the modified fibroin according to the second embodiment, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and more preferably 10% or less. It is even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX described below.

The method of calculating z / w (%) will be described in more detail. First, the amino acid sequence consisting of XGX is extracted from all the REPs contained in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Further, for example, when X (center X) contained in two XGXs exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). be). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w (%) in naturally-derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Among all the extracted fibroin, the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less, which contains the domain sequence represented by the formula (I): [(A) _n motif-REP] _m . From the amino acid sequence of naturally derived fibroin, z / w (%) was calculated by the above-mentioned calculation method. The results are shown in FIG. The horizontal axis of FIG. 3 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 3, the z / w (%) in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).

In the modified fibroin according to the second embodiment, z / w (%) is preferably 50.9% or more, more preferably 56.1% or more, and 58.7% or more. Is even more preferable, 70% or more is even more preferable, and 80% or more is even more preferable. The upper limit of z / w (%) is not particularly limited, but may be, for example, 95% or less.

The modified fibroin according to the second embodiment is prepared, for example, by substituting at least a part of the base sequence encoding the glycine residue from the cloned naturally occurring fibroin gene sequence to encode another amino acid residue. It can be obtained by modifying it. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be replaced so that z / w (%) is 50.9% or more. good. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

The above-mentioned other amino acid residues are not particularly limited as long as they are amino acid residues other than glycine residues, but are valine (V) residue, leucine (L) residue, isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residues and glutamate (E) residues are preferred, with valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) Residues are more preferred, and Glutamine (Q) residues are even more preferred.

As a more specific example of the modified fibroin according to the second embodiment, (2-i) the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or the (2-ii) sequence. Amino acids having sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more with the amino acid sequence represented by No. 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Modified fibroins, including sequences, can be mentioned.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 8 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 6, which corresponds to naturally occurring fibroin. In the amino acid sequence shown in SEQ ID NO: 9, every other (A) _n motif is deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 10, two alanine residues are inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 9, and a part of glutamine (Q) residue is further added. It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have almost the same molecular weight as that of SEQ ID NO: 9. The amino acid sequence shown by SEQ ID NO: 11 is a region of 20 domain sequences existing in the amino acid sequence shown by SEQ ID NO: 10 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w (%) in the amino acid sequence shown in SEQ ID NO: 6 (corresponding to naturally occurring fibroin) is 46.8%. The values of z / w (%) in the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence shown in SEQ ID NO: 11 are 58. 7%, 70.1%, 66.1% and 70.0%. Further, the values of x / y (%) in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown in SEQ ID NOs: 6, 8, 9, 10 and 11 are 15.0%, respectively. 1,5.0%, 93.4%, 92.7% and 89.3%.

The modified fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

The modified fibroin of (2-ii) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Contains an amino acid sequence having 95% or more sequence identity. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . The sequence identity is preferably 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more.

The modified fibroin of (2-ii) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Has 95% or more sequence identity, and the total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in REP is z, and the above domain. When the total number of amino acid residues of REP in the sequence is w, z / w (%) is preferably 50.9% or more.

The modified fibroin described above may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of modified fibroin.

As the tag sequence, for example, an affinity tag using specific affinity (binding, affinity) with other molecules can be mentioned. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chromatography) is performed. Can be used for. As a specific example of the tag sequence, for example, the amino acid sequence shown in SEQ ID NO: 21 (amino acid sequence including His tag) can be mentioned.

In addition, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. By utilizing the epitope tag, the modified fibroin can be easily purified with high specificity.

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the modified fibroin comprising a tag sequence, (2-iii) the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, or (2-iv) SEQ ID NO: 14, Includes an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. , Modified fibroin.

The amino acid sequences represented by SEQ ID NOs: 12, 13, 14, 15, 16 and 17 are the amino acids represented by SEQ ID NO: 21 at the N-terminal of the amino acid sequences represented by SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively. It is the one to which the sequence (including the His tag) is added.

The modified fibroin of (2-iii) may consist of the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

The modified fibroin of (2-iv) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. Contains an amino acid sequence having 95% or more sequence identity. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . The sequence identity is preferably 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more.

The modified fibroin of (2-iv) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. Has 95% or more sequence identity, and the total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in REP is z, and the above domain. When the total number of amino acid residues of REP in the sequence is w, z / w (%) is preferably 50.9% or more.

The modified fibroin described above may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The modified fibroin according to the third embodiment has an amino acid sequence whose domain sequence has a reduced content of (A) _n motif as compared with naturally occurring fibroin. It can be said that the domain sequence of the modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs as compared with the naturally occurring fibroin.

The modified fibroin according to the third embodiment may have an amino acid sequence corresponding to the deletion of (A) _n motif by 10 to 40% from naturally occurring fibroin.

The modified fibroin according to the third embodiment has a domain sequence of 1 to 3 (A) _n motifs from at least the N-terminal side toward the C-terminal side as compared with the naturally occurring fibroin (1). A) It may have an amino acid sequence corresponding to the deletion of the _n -motif.

The modified fibroin according to the third embodiment has a domain sequence of at least two consecutive deletions of the (A) _n motif from the N-terminal side to the C-terminal side as compared with the naturally occurring fibroin, and the deletion of the n motif. It may have an amino acid sequence corresponding to the deletion of one (A) _n motif repeated in this order.

The modified fibroin according to the third embodiment has an amino acid sequence corresponding to the deletion of the (A) _n motif at least every other two domain sequences from the N-terminal side to the C-terminal side. There may be.

The modified fibroin according to the third embodiment contains a domain sequence represented by the formula (I): [(A) _n motif-REP] _m , and is adjacent to two adjacent cells from the N-terminal side to the C-terminal side. When the number of amino acid residues of the REP of the [(A) _n motif-REP] unit is sequentially compared and the number of amino acid residues of the REP having a small number of amino acid residues is 1, the number of amino acid residues of the other REP is increased. Let x be the maximum value of the sum of the amino acid residues of two adjacent [(A) _n motif-REP] units having a ratio of 1.8 to 11.3, and let x be the total amino acid residues of the domain sequence. When the number is y, it may have an amino acid sequence in which x / y (%) is 20% or more, or it may have an amino acid sequence in which x / y (%) is 50% or more. (A) The number of alanine residues with respect to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y (%) will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residue)-(A) _n motif-second REP (100 amino acid residue)-(A) _n . Motif-Third REP (10 amino acid residues)-(A) _n Motif-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) It has an arrangement called _n motifs.

Two adjacent [(A) _n -motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, there may be a [(A) _n motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) _n motif-REP] units is compared. The comparison is made by finding the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparison between the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), the 5th REP is assumed to be 1 when the 4th REP having a smaller number of amino acid residues is 1. The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is 1.8 to _11.3 . Shown by a solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than _11.3 . Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of (A) _n motif. be.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximum is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the modified fibroin according to the third embodiment, x / y (%) is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, and 70%. The above is even more preferable, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x / y (%) is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y (%) is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4. The x / y (%) is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, the x / y (%) is 75.9% or more. When the jagged ratio is 1: 1.9 to 4.1, x / y (%) is preferably 64.2% or more.

When the modified fibroin according to the third embodiment is a modified fibroin in which at least 7 of (A) _n motifs present in a plurality of (A) n motifs are composed of only alanine residues, x / y (%) is 46. .4% or more is preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further preferable, and 70% or more is further more preferable. Further, it is preferable, and it is particularly preferable that it is 80% or more. The upper limit of x / y (%) is not particularly limited and may be 100% or less.

Here, x / y (%) in naturally-derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroins, from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula (I): [(A) _n motif-REP] _m , x by the above calculation method. / Y (%) was calculated. The results when the jagged ratio is 1: 1.9 to 4.1 are shown in FIG.

The horizontal axis of FIG. 2 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the x / y (%) of naturally occurring fibroin is less than 64.2% (highest, 64.14%).

The modified fibroin according to the third embodiment is, for example, a sequence encoding the (A) _n motif so that x / y (%) is 64.2% or more from the cloned gene sequence of naturally derived fibroin. It can be obtained by deleting one or more. Further, for example, an amino acid sequence corresponding to the deletion of one or a plurality of (A) _n motifs so that x / y (%) is 64.2% or more is designed from the amino acid sequence of naturally occurring fibroin. However, it can also be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the deletion of (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the modified fibroin according to the third embodiment, (3-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or the (3-ii) sequence. Amino acids having sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more with the amino acid sequence represented by No. 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Modified fibroins, including sequences, can be mentioned.

The modified fibroin of (3-i) will be described. The amino acid sequence shown in SEQ ID NO: 7 is obtained by deleting every other (A) _n motif from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, which corresponds to naturally occurring fibroin. Further, one [(A) _n motif-REP] is inserted before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 9 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 7. In the amino acid sequence shown in SEQ ID NO: 10, two alanine residues are inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 9, and a part of glutamine (Q) residue is further added. It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have almost the same molecular weight as that of SEQ ID NO: 9. The amino acid sequence shown by SEQ ID NO: 11 is a region of 20 domain sequences existing in the amino acid sequence shown by SEQ ID NO: 10 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

The value of x / y (%) in the Giza ratio 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 6 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y (%) in the amino acid sequence shown by SEQ ID NO: 7 and the amino acid sequence shown by SEQ ID NO: 9 is 93.4%. The value of x / y (%) in the amino acid sequence represented by SEQ ID NO: 10 is 92.7%. The value of x / y (%) in the amino acid sequence shown in SEQ ID NO: 11 is 89.3%. The values of x / y (%) in the amino acid sequences set forth in SEQ ID NOs: 6, 7, 9, 10 and 11-12 are 46.8%, 56.2%, 70.1%, 66.1% and 66.1%, respectively. It is 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

The modified fibroin of (3-ii) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Contains an amino acid sequence having 95% or more sequence identity. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . The sequence identity is 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more.

The modified fibroin of (3-ii) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Has 95% or more sequence identity, and sequentially compares the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units from the N-terminal side to the C-terminal side. When the number of amino acid residues in a REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (the jagged ratio is 1: 1.8 to 11.3). ), When the maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n motif-REP] units is x and the total number of amino acid residues of the domain sequence is y. It is preferable that x / y (%) is 64.2% or more.

The above-mentioned modified fibroin may contain the above-mentioned tag sequence at either or both of the N-terminal and the C-terminal.

As a more specific example of the modified fibroin containing the tag sequence, (3-iii) the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, or (3-iv) SEQ ID NO: 13, Includes an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. , Modified fibroin can be mentioned.

The amino acid sequences represented by SEQ ID NOs: 12, 13, 14, 15, 16 and 17 are the amino acids represented by SEQ ID NO: 21 at the N-terminal of the amino acid sequences represented by SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively. It is the one to which the sequence (including the His tag) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

The modified fibroin of (3-iv) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. Contains an amino acid sequence having 95% or more sequence identity. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by the formula (I): [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) is 80% or more, preferably 85% or more, more preferably 90% or more, most preferably the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. Has 95% or more sequence identity, and sequentially compares the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units from the N-terminal side to the C-terminal side. , When the number of amino acid residues of REP with a small number of amino acid residues is 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3. Two adjacent [(A) _n motifs- When the maximum value of the total value obtained by adding the number of amino acid residues in the REP] unit is x and the total number of amino acid residues in the domain sequence is y, x / y (%) is 64.2% or more. Is preferable.

The modified fibroin described above may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The modified fibroin according to the fourth embodiment has a domain sequence in which the content of (A) _n motif is reduced as compared with the naturally occurring fibroin, and the content of glycine residue is reduced. It has an amino acid sequence. The domain sequence of the modified fibroin lacks at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with an amino acid residue. That is, it is a modified fibroin having the characteristics of the modified fibroin according to the second embodiment described above and the modified fibroin according to the third embodiment. Specific embodiments and the like are as described in the modified fibroin according to the second and third embodiments.

As a more specific example of the modified fibroin according to the fourth embodiment, (4-i) the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, (4-ii) SEQ ID NO: 9, SEQ ID NO: 10 or a modified fibroin containing an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11. Can be done. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 are as described above.

<Protein manufacturing method>
The protein according to this embodiment is the nucleic acid, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing.

The method for producing the nucleic acid encoding the protein is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. can do. The chemical synthesis method of nucleic acid is not particularly limited, and for example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) based on the amino acid sequence information of the protein obtained from the NCBI web database or the like. Genes can be chemically synthesized by ligating automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. May be good.

The regulatory sequence is a sequence that controls the expression of modified fibroin in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding a protein can be transcribed is preferably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacillus, Corinebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satillas and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Phyllum and the like. Examples of the microorganism belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, as a vector for introducing a nucleic acid encoding a protein, for example, pBTrp2 (manufactured by Berliner Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569) and the like can be mentioned.

Examples of eukaryotic hosts include yeast and filamentous fungi (molds and the like). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of the filamentous fungus include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

When a eukaryote is used as a host, examples of the vector into which the nucleic acid encoding the modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like can be mentioned.

As a method for expressing nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates containing them such as honey, starch and starch hydrolysate, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used. As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted by using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

Further, during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The expressed protein can be isolated and purified by a commonly used method. For example, when the protein is expressed in a lysed state in the cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then an ultrasonic crusher, a French press, or manton gaulin. Crush the host cells with a homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting out method using sulfur cheaper, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -cepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis, etc. Purified preparations can be obtained by using the above methods alone or in combination.

When the protein is expressed by forming an insoluble substance in the cell, the host cell is similarly recovered, crushed, and centrifuged to recover the insoluble protein as a precipitate fraction. The insoluble form of the recovered protein can be solubilized with a protein denaturing agent. After the operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant and using the same isolation and purification method as described above from the culture supernatant.

(2) Polypeptide and Polyamino Acid Having β-Sheet Structure In the composite molding composition of the present invention, the above-mentioned embodiment contains the polypeptide or polyamino acid having the β-sheet structure. In this case, examples of polyamino acids include polyalanine.

The polyalanine can be selected from liner-type polyalanine (L-polyAla) or telechelic-type polyalanine (T-polyAla). Liner-type polyalanine can be produced by utilizing an enzymatic chemical polymerization method, and can be produced and used based on known literature (Baker P J et al., Biomacromolecules 2012, 13, 947-951). In addition, the telechelic polyalanine can be produced by using an enzymatic chemical polymerization method as described in the following examples, and is also described in Tsuchiya K et al., Macromol. Biosci. 2016, 1001-1008, and by citation. Incorporated herein.

(3) Production of Composite Molded Composition Containing Fibroin and Polypeptide and / or Polyamino Acid Having β-Sheet Structure In the present specification, "polypeptide having β-sheet structure" is defined in its two-dimensional structure. , Any polypeptide as long as it has a β-sheet structure, including natural and artificially produced polypeptides, preferably artificially produced polypeptides, more preferably: Refers to a polypeptide containing the sequence of polyamino acids.

As used herein, the term "polyamino acid" refers to a high molecular weight amino acid polymer obtained by a one-step polymerization reaction using an amino acid or a derivative thereof as a monomer.

Further, the polyamino acid having a β-sheet structure is an artificially produced polymer of L- or D-amino acid, and refers to any polyamino acid having a β-sheet structure in its secondary structure, preferably poly. Homopolyamino acids selected from alanine, polyphenylalanine, polycysteine, polyvaline, polyleucine, polyisoleucine, polytyrosine, polytryptophan, polyglutamine, polymethionine and their derivatives, and homopolyamino acids thereof and their derivatives. Not only the derivatives thereof, but also amino acids such as alanine, phenylalanine, cysteine, valine, leucine, isoleucine, tyrosine, tryptophan, glutamine, methionine, and a plurality of kinds selected from those derivatives, preferably 2 to 5 kinds, more preferably. Contains polyamino acids consisting of a copolymer of 2-4, most preferably 2 or 3 amino acids. These polyamino acids can be produced by known methods such as, for example, chemical enzyme polymerization methods (Numata K. et al., Polymer Journal, 2015; 47: 537-545, and Baker JP et al., Biomacromolecules 2012, 13, 947- 951 etc.). The degree of polymerization of the polyamino acid is in the range of 2 to 100, preferably in the range of 5 to 50, more preferably in the range of 10 to 30, and most preferably in the range of 10 to 20.

As used herein, the structures of polyamino acids, polypeptides and proteins are described in three-letter or one-letter notation of amino acids well known to those of skill in the art. In the specification, amino acids are L-form unless otherwise specified.

The nanogranular structure, which is one of the structures constituting silk derived from spider silk and is a small granular structure constituting nanofibrils, has a granular morphology having a high aspect ratio. A structure containing a large amount of glycine and alanine, which is characteristic of silk, and containing a β-sheet structure or a polypeptide as a main component. Nephila edulis spider silk has been reported to have a β-sheet structure of 17% ± 4% (Ling S et al., Biomacromolecules, 2011, 12, 3344-3349). In addition, 45-65% of the strongest spider traction yarns have been reported to be β-sheet domains (Vollrath F et al., Polymer, 2009, 50, 5623-5632).

Therefore, in the composite molding composition of the present invention, a dope solution prepared by mixing and dissolving a polypeptide or polyamino acid containing a large amount of β-sheet structure in a fibroin-like protein as a raw material is prepared, and a composite fiber, a composite film, a composite gel, etc. It can be manufactured by molding a composite porous body, a composite particle, a composite molded body, or the like.

In the case of producing a composite film, a method for producing a film using a fibroin-derived protein as a raw material is described in International Publication WO2014 / 103799, and the film can be basically produced according to this. When producing a composite fiber, a method of spinning the fiber from a fibroin-derived protein is described in International Publication WO2012 / 165476, and the fiber can be basically produced according to this method. When producing a composite gel, WO2014 / 175177 describes a method for producing a gel from a fibroin-derived protein, and the gel can be basically produced according to this method. Further, in the case of producing a composite porous body, a method for producing a porous body from a fibroin-derived protein is described in International Publication WO2014 / 175178, and it can be basically produced according to this method. Further, in the case of producing composite particles, a method for producing particles from fibroin-derived protein is described in International Publication WO2014 / 175179, and the particles can be basically produced according to this method. Further, in the case of producing a composite molded article, the specification of Japanese Patent Application No. 2015-185777 describes a method for producing a molded article from a fibroin-derived protein, and the article can be basically produced according to this method.

When the composite molding composition is a composite film produced by using the dope solution, the coating method is generally known in the art, for example, casting method, spin coating method, dipping method, spray coating. A method such as a method, an electropolymerization method, a vapor deposition method, a vapor deposition polymerization method, a brush coating method, a blade coating method, a roller coating method, a gravure coating method, and a roll-to-roll method can be used.

Further, in the case of spinning using a dope solution in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein, the pH and salt of the dope solution are applied while applying a breaking stress in the spinning step. Fibroin-derived composite fibers can be produced by changing the environment-related factors of fibroin-derived polypeptide fibers such as spider silk, such as type and concentration, humidity or water concentration. A fibroin-derived composite fiber can be produced by changing one or more of these elements or by changing a combination of a plurality of elements. Specific examples of the method for producing a composite fiber derived from fiproin of the present invention include known spinning methods such as a wet spinning method, a dry spinning method, a dry wet spinning method, and a melt spinning method.

In the embodiment of the present invention, when the wet spinning method or the dry wet spinning method is used, a coagulant is added to the dope solution while applying shear stress in the fiber axis direction, or the dope solution is contained in a solvent containing the coagulant. By injecting, extruding or immersing the fiber, a polypeptide fiber derived from fibroin can be produced.

The coagulant in the wet prevention method or the dry-wet spinning method is not particularly limited as long as it can remove the solvent in which the fibroin-derived protein or the like is dissolved from the dope solution (also referred to as desolvent). For example, as the coagulant, a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, or acetone may be used. Further, an aqueous solution containing an inorganic salt may be used. The inorganic salt aqueous solution is preferably a weakly acidic to acidic solvent.

For example, in the case of producing a composite molded product, a composition containing a protein (containing only protein or other components) is introduced into a mold of a pressure molding machine, and then the mold is heated and the composition is formed. Pressurize against. Heating and pressurization are continued under a predetermined pressure until the protein powder reaches a predetermined temperature to obtain a heated and pressurized composition. Next, the temperature of the mold is lowered by using a cooler (for example, a spot cooler), and when the composition reaches a predetermined temperature, the contents are taken out to obtain a molded product. The heating is preferably performed at 80 to 300 ° C, more preferably 100 to 180 ° C, still more preferably 100 to 130 ° C. Pressurization is preferably performed at 5 kN or more, more preferably 10 kN or more, still more preferably 20 kN or more. Further, the time (heat retention condition) for continuing the treatment under the predetermined heating and pressurizing condition is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, still more preferably 5 to 30 minutes.

For example, in the case of producing a resin, a dope solution in which the composition of the present invention is dissolved or suspended in a solvent is prepared, and the protein of the dope solution is insolubilized by a method well known to those skilled in the art. , The resin of the present invention can be produced. Examples of the solvent include water, a polar organic solvent, or a mixed solvent thereof. Examples of the method for insolubilizing the protein of the present invention include distilling off the solvent of the dope solution, changing the type and / or concentration of the salt in the solvent, changing the ionic strength or salt concentration, and / or changing the pH.

In the present specification, the "dope solution" is a mixture of a fibroin-derived protein which is a raw material for producing a composite molding composition such as a composite fiber or a composite film, and a polypeptide having a β-sheet structure and / or a polyamino acid. It means a dissolved solution.

Examples of the polar organic solvent used in the above-mentioned dope solution include dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide or 1,1,1,3,3,3-hexafluoro-2-propanol. You can choose from (HFIP), formic acid, or mixtures thereof, but are not limited to these.

Further, the dope solution may further contain an inorganic salt. Inorganic salts can serve as protein lysis promoters. Examples of the inorganic salt include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, and thiocyanate. Specific examples of the inorganic salt include aluminum phosphate, lithium carbonate, aluminum carbonate, aluminum sulfate, aluminum fluoride, ferric acetate, aluminum acetate, zinc hydroxide, magnesium hydroxide, ferrous hydroxide, and manganese hydroxide. , Chromium hydroxide, ferric hydroxide, aluminum hydroxide, nickel chloride, cobalt chloride, zinc chloride, ferrous chloride, manganese chloride, chromium chloride, ferric chloride, aluminum chloride, lithium nitrate, strontium nitrate, nitrate Nickel, calcium nitrate, cobalt nitrate, zinc nitrate, magnesium nitrate, ferrous nitrate, manganese nitrate, chromium nitrate, ferric nitrate, aluminum nitrate, lithium bromide, barium bromide, strontium bromide, nickel bromide, odor Calcium bromide, cobalt bromide, zinc bromide, magnesium bromide, ferrous bromide, manganese bromide, chromium bromide, ferric bromide, aluminum bromide, barium chlorate, strontium chlorate, nickel chlorate , Calcium chlorate, cobalt chlorate, zinc chlorate, magnesium chlorate, ferrous chlorate, manganese chlorate, chromium chlorate, ferric chlorate, aluminum chlorate, rubidium iodide, sodium iodide, yo Copper oxide, lithium iodide, barium iodide, strontium iodide, nickel iodide, calcium iodide, cobalt iodide, zinc iodide, magnesium iodide, ferric iodide, manganese iodide, chromium iodide, iodine Ferric iron, aluminum iodide, sodium perchlorate, lead perchlorate, copper perchlorate, lithium perchlorate, barium perchlorate, strontium perchlorate, nickel perchlorate, calcium perchlorate, excess Cobalt chlorate, zinc perchlorate, magnesium perchlorate, ferrous chlorate, manganese perchlorate, chromium perchlorate, ferric perchlorate, aluminum perchlorate, potassium thiocyanate, sodium thiocyanate , Lead thiocyanate, copper thiocyanate, lithium thiocyanate, barium thiocyanate, strontium thiocyanate, nickel thiocyanate, calcium thiocyanate, cobalt thiocyanate, zinc thiocyanate, magnesium thiocyanate, ferric thiosianate, manganese thiosianate , Chromium thiosicate, ferric thiosicate, aluminum thiocyanate, ammonium cyanate, cesium cyanate, rubidium cyanate, potassium cyanate, sodium cyanate, lead cyanate, copper cyanate, lithium cyanate, barium cyanate , Cyanic acid Examples thereof include strontium, nickel cyanate, calcium cyanate, cobalt cyanate, zinc cyanate, magnesium cyanate, ferrous acid, manganese cyanate, chromium cyanate, ferric cyanate, and aluminum cyanate. At least one of these inorganic salts may be added to the solvent.

The amount of the inorganic salt contained in the dope solution is not particularly limited, and is appropriately determined according to the type of the inorganic salt, the amount of fibroin-derived protein, and the like. The amount of the inorganic salt is, for example, 1.0 part by mass or more, 5.0 parts by mass or more, 9.0 parts by mass or more, 15 parts by mass or more, and 20 parts by mass or more with respect to 100 parts by mass of the total amount of protein. May be. Further, the amount of the inorganic salt may be, for example, 40 parts by mass or less, 35 parts by mass or less, and 30 parts by mass or less with respect to 100 parts by mass of the total amount of protein.

In the present specification, the amount of the polypeptide and / or the polyamino acid added to the fibroin-derived protein is the total weight of the polypeptide and / or the polyamino acid to be added to the fibroin-derived protein and / or the polyamino acid. Expressed as a percentage of weight. For example, when described as "polyAla 5 wt% composite molding composition", a composite molding composition containing (fibroin-derived protein) :( polypeptide and / or polyamino acid) in a weight ratio of 95: 5. Represents an object. In some cases, it is expressed as a ratio to mass.

The composition containing fibroin and a polypeptide having a β-sheet structure and / or a polyamino acid of the present invention is, for example, an aqueous solution or water of a polypeptide having a β-sheet structure and / or a polyamino acid in a dope solution in which fibroin is dissolved. It can be produced by adding a suspension, mixing, molding, and drying. The blending ratio of the polypeptide having a β-sheet structure and / or the polyamino acid is in the range of 0.1 to 50.0%, preferably in the range of 0.5 to 30.0%, and more preferably 1 It is in the range of ~ 10.0%, most preferably in the range of 1-5%.

When polyalanine is used as the polyamino acid having a β-sheet structure, the degree of polymerization of polyalanine is in the range of 2 to 100, preferably in the range of 3 to 50, and more preferably in the range of 10 to 30. It is most preferably in the range of 10 to 20. The compounding ratio of polyalanine is in the range of 0.1 to 30.0%, preferably in the range of 0.5 to 20.0%, more preferably in the range of 1.0 to 10.0%, and most preferably in the range of 3.0 to 5.0%. Is.

1-2 . Stretched Composite Molding Composition Another embodiment of the present invention is a stretched composite molding composition . The stretched composite composition is produced by further including a stretching step in the manufacturing step of the composite molding composition, and brings about better physical characteristics as compared with the non-stretched composite molding composition. be able to.

Specifically, the stretched composite molding composition of the present embodiment is a stretched composite molding composition in which a polypeptide having a β-sheet structure and / or a polyamino acid is blended with a fibroin-derived protein.
The composite molding composition is selected from fibers or films.
(i) Preparation of a dope of fibroin-derived protein and polypeptide and / or polyamino acid having a β-sheet structure,
(ii) A step of molding a composite molding composition from the dope solution,
moreover,
(iii) The step of stretching the composite molding composition obtained in (ii) above in a solvent and drying it.
It is a stretched composite molding composition manufactured by the manufacturing method including.

By adding a stretching step, physical properties such as tensile strength, strain and / or toughness are further improved . Examples of the stretching step include wet heat stretching and dry heat stretching.

Wet and heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, in an organic solvent, or in steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In moist heat stretching, the unstretched fiber or film ( or stretched fiber or film) can be stretched, for example, 1 to 10 times, and preferably 1 to 4 times.

Dry heat stretching can be performed using an electric tube furnace, a dry heat plate, or the like. The temperature may be, for example, 140 to 270 ° C, preferably 160 to 230 ° C. In dry heat stretching, the unstretched fiber or film ( or stretched fiber or film) can be stretched, for example, 0.5 to 8 times, and preferably 1 to 4 times.

Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first step stretching is performed by moist heat stretching, the second step stretching is performed by Kanetsu stretching, or the first step stretching is performed by moist heat stretching, the second step stretching is performed by moist heat stretching, and the third step stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.

From these composite molding compositions of the present invention, for example, threads, mattresses, non-layouts, meshes, nets and the like using them can be manufactured. Taking advantage of the excellent properties of these composite molded compositions such as heat resistance, high tensile strength, toughness and / or extensibility (elongation), for example, high impact resistance of bulletproof vests, parachutes, automobile bodies, etc. It can be used to manufacture the required materials. In addition, it can be used as a medical material such as a wound closure material, a suture, an adhesive plaster, and a scaffold material for regenerative medicine utilizing high strength, high extensibility and high toughness, and biodegradability and biocompatibility.

2. 2. Method for Producing Composite Molded Composition of the Present Invention 2-1. Method for Producing Non-stretched Composite Molded Composition Another embodiment of the present invention is a method for producing the composite molded composition. More specifically, the present invention is a method for producing a composite molding composition selected from fibers, films, and gels, which comprises a fibroin-derived protein containing a polypeptide having a β-sheet structure and / or a polyamino acid. ,
(i) A step of preparing a dope solution in which a fibroin-derived protein and a polypeptide having a β-sheet structure and / or a polyamino acid are dissolved.
(ii) A step of producing the composite molding composition by spinning or stretching the dope solution.
It is a manufacturing method including.

As described above, for example, fibroin-derived proteins, natural fibroin proteins are commercially available and can be obtained and used in the present invention. Further, as described above, a fibroin-derived protein produced by a microorganism into which DNA artificially prepared by a gene recombination method has been introduced and modified by isolation and purification can be used. Fibroin is obtained by these methods, and separately known methods such as, for example, a chemical enzyme polymerization method (Numata K. et al., Polymer Journal, 2015; 47: 537-545, and Baker J. P. et al., Biomacromolecules 2012, 13 , 947-951, etc.) to produce a polypeptide and / or a polyamino acid having a β-sheet structure, mix the polypeptide and / or an aqueous suspension of the polyamino acid with a dope solution containing a fibroin-derived protein, and then dry, etc. The composition of the present invention can be produced by insolubilizing with.

As the method for insolubilizing, other than the method for drying, for example, the same method as described in the method for producing a composite fiber, specifically, a change in the type and / or concentration of a salt in a solvent, and an ionic strength. Alternatively, a method such as a change in salt concentration and / or a change in pH can be used.

Further, as for the material other than the fibroin-derived protein, the production method of the present invention can be carried out by using various materials described in detail in the above composite molding composition and using them in the usage mode described in detail above. ..

2-2 . Method for Producing Stretched Composite Molded Composition Another embodiment of the present invention is a method for producing a stretched composite molded composition.
(i) A step of stretching the composite molding composition produced by the above-mentioned production method in a solvent.
as well as,
(ii) A step of drying the stretched composite molding composition,
It is a manufacturing method including.

By adding a stretching step, physical properties such as tensile strength, strain and / or toughness are further improved . Examples of the stretching step include wet heat stretching and dry heat stretching.

Wet and heat stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, in an organic solvent, or in steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In moist heat stretching, the non-stretched fiber or film ( or stretched fiber or film) can be stretched, for example, 1 to 10 times, and preferably 1 to 4 times.

Dry heat stretching can be performed using an electric tube furnace, a dry heat plate, or the like. The temperature may be, for example, 140 to 270 ° C, preferably 160 to 230 ° C. In dry heat stretching, the unstretched fiber or film ( or stretched fiber or film) can be stretched, for example, 0.5 to 8 times, and preferably 1 to 4 times.

Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first-stage stretching is performed by moist heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by moist heat stretching, the second stage stretching is performed by moist heat stretching, and the third stage stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.

The composite molded composition produced by these production methods of the present invention can further produce, for example, threads, mattresses, non-layouts, meshes, nets and the like using the composite molding compositions. Taking advantage of the excellent properties of these composite molding compositions such as heat resistance, high tensile strength, toughness and / or extensibility, for example, for materials requiring high impact resistance such as bulletproof vests, parachutes, and automobile bodies. Can be used for manufacturing. In addition, it can be used as a medical material such as a wound closure material, a suture, an adhesive plaster, and a scaffold material for regenerative medicine utilizing high strength, high extensibility and high toughness, and biodegradability and biocompatibility.

3. 3. Method for Improving Physical Properties of Composite Molding Composition of the Present Invention Another preferred embodiment of the present invention is selected from tensile strength, toughness, extensibility and the like of a composite molding composition composed of a polypeptide such as fibroin. A way to improve at least one physical property. Examples of the composite molding composition include a composite film made of a polypeptide, a composite fiber, a composite gel, a composite porous body, a composite particle, a composite molded body, and the like.

As a method for improving the physical properties of a composite molding composition, for example, a polyamino acid having a β sheet such as polyalanine is blended with a molding composition raw material such as a resin, a film and a fiber to prepare a composite molding composition. according to. More specifically, for example, a polypeptide having a β-sheet structure in a dope solution in which a raw material such as fibroin is dissolved in order to produce a composite molding composition such as a composite film, a composite fiber, a composite gel and a composite resin. And / or by adding the polyamino acid and mixing, for example, distilling off the solvent to insolubilize the polypeptide, as compared with the polypeptide having a β-sheet structure and / or the molding composition without the addition of the polyamino acid. , A composite molding composition having improved physical properties can be obtained.

As used herein, improving the physical properties of a composite molding composition means, for example, obtaining a commercially available tensile tester, performing a tensile strength test, and determining the critical stress when the test sample breaks. , When the value of this critical stress is higher than that of the sample to be compared, it means that the tensile strength is improved. Similarly, toughness is improved when the test sample is measured with a tensile tester and the area value of the curve in the stress-strain curve shows a higher value than the area value of the sample to be compared. Means to improve. Further, to improve the extensibility, the tensile test of the test sample is similarly performed with a tensile tester to determine the strain (stretching ratio) when the test sample breaks, and the strain (stretching ratio) when the test sample breaks is measured with the sample to be compared under the same conditions. It means that the elongation rate at the time of breaking of the test sample is improved when the stretch rate at the time of breaking shows a higher value than the strain at the time of breaking obtained.

Examples of the polyamino acid used in the method of the present invention include polyamino acids that easily form a β-sheet structure, and more specifically, polyalanine, polycysteine, and the like. These polyamino acids can be produced by known methods such as chemical enzyme synthesis (Numata K. et al., Polymer Journal, 2015; 47: 537-545, and Baker J.P. et al., Biomacromolecules 2012, 13, 947-951, etc. ).

For example, it is carried out by blending polyalanine with a molding composition raw material composed of a fibroin-derived polypeptide. When improving the physical properties of the composite molded composition, the degree of polymerization is in the range of 2 to 100, preferably in the range of 5 to 50, more preferably in the range of 10 to 30, and most preferably in the range of 10 to 10. Polyalanine in the range of 20 is blended in a proportion of 0.1 to 30.0%, preferably 0.5 to 20.0%, more preferably 1.0 to 10.0%, and most preferably 3.0 to 5.0%. Add to the dope solution.

By using the method of the present invention, it is possible to manufacture threads, mattresses, non-layouts, meshes, nets and the like having improved physical properties. Taking advantage of the excellent properties of these composite molding compositions such as heat resistance, high tensile strength, toughness and / or extensibility, for example, for materials requiring high impact resistance such as bulletproof vests, parachutes, and automobile bodies. Can be used for manufacturing. It can also be used for manufacturing medical materials such as wound closure materials, sutures, adhesive plasters, and scaffolding materials for regenerative medicine, which utilize high strength, high extensibility and high toughness, and biodegradability and biocompatibility.

All documents referred to herein are incorporated herein by reference in their entirety.

The embodiments of the present invention described below are for illustration purposes only and do not limit the technical scope of the present invention. The technical scope of the invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention may be made on condition that the gist of the present invention is not deviated.

1. Manufacture of composite molded film containing polyalanine (L-polyAla or T-polyAla) in natural silk moth silk fibroin protein or modified spider silk fibroin protein and evaluation of its physical properties. Production of composite molded film containing polyalanine in natural silk moth silk fibroin protein Synthesize polyAla (L-polyAla or T-polyAla) by the method described below to combine with natural silk moth fibroin protein (Bombix mori). Prepare a dope solution prepared by dissolving polyalanine (5 wt% or 10 wt% of L-polyAla or 1 wt% or 2.5 wt% of T-polyAla) produced by the following method, and use the casting method from the dope solution. A composite molded film was manufactured, a tension deformation test was performed, and changes in physical properties were evaluated.

The tension deformation test was carried out on a small tabletop tester (EZ-LX type, Shimadzu Corporation, Kyoto). The initial sample length was 15.0 mm and was stretched at a constant rate of 0.5 mm / min. The results were recorded and analyzed by TRAPEZIUM (ver. 1.3.0, Shimadzu Corporation, Kyoto). The measurements were performed at 58% humidity and room temperature.

2. 2. Manufacture of polyAla (L-polyAla or T-polyAla)
(1) Synthesis of telechelic polyalanine (T-polyAla)
(i) Synthesis using a leucine initiator (Leu-initiator)
(a) Synthesis of leucine initiator The synthesis of telechelic polyalanine using a leucine initiator was produced by a chemical enzyme synthesis method using papain. Specifically, L-leucine ethyl ester hydrochloride (6.07 g), triethylamine (9.2 mL) and diethyl ether (100 mL) were added to a flask under a nitrogen environment at 0 ° C., and succinyl chloride (1.7 mL) was added to this solution. Diethyl ether solution (50 mL) was added dropwise over 30 minutes, and the mixture was stirred at 0 ° C. for 2 hours. After warming the reaction solution to room temperature, water was added and the aqueous layer was extracted with diethyl ether. The organic layer was dried over sodium sulfate and then concentrated. The crude product was vacuum dried and recrystallized from hexane / ethyl acetate = 5: 1 (v / v) to obtain 3.57 g (yield 60%) of yellow needle-like crystals of the leucine initiator (Leu-initiator).

(b) Synthesis of telechelic polyalanine (T-polyAla) by chemical enzyme synthesis method Alanine ethyl ester hydrochloride (0.645 g), the above-mentioned leucine initiator (0.080 g), phosphate buffer (2 mL, 1 M, pH 8.0) and Ethanol (1 mL) was added to the glass tube and stirred at 40 ° C. until all substrates were dissolved. A solution of papain (0.300 g) in phosphate buffer (2.2 mL) was poured into this solution at one time. The final concentrations of alanine and papain were 0.7 M and 50 mg / mL, respectively. The mixture was stirred at 40 ° C. for 6 hours. The stirred mixture was cooled to room temperature and precipitated by centrifugation (7000 rpm, 4 ° C., 10 minutes). The crude precipitate was washed twice with deionized water and lyophilized to give 0.071 g of a white solid oligopeptide.

(ii) Synthesis of telechelic polyalanine using bis (alanine ethyl ester) initiator
(a) Synthesis of bis (alanine ethyl ester) initiator For synthesis using the bis (alanine ethyl ester) initiator, alanine ethyl ester hydrochloride (4.76 g), triethylamine (9.2 mL) and chloroform (100) are placed in a 200 mL flask. mL) was added, and a solution of succinyl chloride (1.7 mL) in chloroform (50 mL) was added dropwise to this mixed solution at 0 ° C. under a nitrogen atmosphere. After stirring this mixture at 0 ° C. for 2 hours, water was added to stop the reaction. The mixture was washed with water, 1 M aqueous sodium hydrogen carbonate solution and brine. The organic layer was dried over sodium sulfate and then concentrated. The crude product was recrystallized from hexane / ethyl acetate to give 3.58 g of white needle-like crystals (yield 76%).
The results of infrared absorption spectrum and NMR spectrum measurement and combustion elemental analysis are as follows.
Infrared (IR) (neat): ν = 3301, 2991, 1729, 1639, 1545, 1356, 1238, 1204, 1168, 1020 cm ^-1.1 H NMR (500 MHz, CDCl ³ _, 25 ° C, ppm): δ6.66 (s, 2H), 4.53 (m, 2H) 4.20 (q, J = 7.1 Hz, 4H), 2.57 (m, 4H), 1.40 (d, J = 7.1 Hz, 6H), 1.28 (t, J = 7.1 Hz, 6H). ¹³ C NMR (125 MHz, CDCl ₃ , 25 ° C): δ 173.16, 171.73, 61.37, 48.18, 31.55, 17.97, 14.04.Anal. Calcd for C ₁₄ H ₂₄ N ₂ O ₆ : C, 53.15; H, 7.65; N, 8.86. Found: C, 53.08; H, 7.61; N, 8.85.

(b) Synthesis of telechelic polyalanine (T-polyAla) by chemical enzyme synthesis method
In a 10 mL glass tube, add alanine ethyl ester hydrochloride (0.922 g), the above bis (alanine ethyl ester) initiator (0.190 g), phosphate buffer (2.0 mL, 1 M, pH 8.0) and tetrahydrofuran (1.0 mL). In addition, the mixture was stirred at 40 ° C. until all substrates were completely dissolved. A phosphate buffer solution (2.0 mL) of papain (0.300 g) was poured at one time. The final concentrations of alanine ethyl ester and papain were 1 M and 50 mg / mL, respectively. The mixture was stirred at 40 ° C. for 6 hours. The stirred mixture was cooled to room temperature and then precipitated by centrifugation (7000 rpm, 4 ° C., 10 minutes). The crude product was washed twice with deionized water and methanol and then lyophilized to give 0.221 g (61%) of white powder.

(2) Production of liner type (linear) polyalanine Liner type (linear) using the same method as telechelic type polyalanine (T-polyAla) except that there is no bis (alanine ethyl ester) initiator. Polyalanine (L-polyAla) was synthesized.

(3) Measurement of average degree of polymerization and average molecular weight of polyalanine The average molecular weight of the manufactured L-polyAla and T-polyAla was measured by NMR (Varian NMR System 500, Varian Medical Systems, Palo Alto, CA, USA). As a result of calculation, the average degree of polymerization of L-polyAla was 5.8 and the average molecular weight was 531 and the average degree of polymerization of T-polyAla was 5.9 and the average molecular weight was 593.

Examples of such synthesis of telechelic polyalanine (T-polyAla) are described in detail in, for example, the literature of Tsuchiya K. et al. (Tsuchiya K. et al., Macromol. Biosci. 2016, 16, 1001-1008). It has been disclosed and the entire content of this disclosure is incorporated herein by reference.

3. 3. Production of modified spider silk fibroin protein and production of composite film containing polyAla As modified spider silk fibroin protein, recombinant spider silk protein ADF3KaiLargeNRSH1; SEQ ID NO: 4 and ADF3Kai_noNR: SEQ ID NO: 18 ) Was produced according to a known method (Japanese Patent Laid-Open No. 2014-129639) and used for producing a composite molded film.

Using the above-mentioned modified spider silk fibroin protein, a composite film of the modified spider silk fibroin protein was produced by a casting method in the same manner as in the production of the above-mentioned natural silk moth fibroin protein composite film.

4. Tension deformation test of composite film A tension deformation test was carried out on the above composite film by the same method as that of the above natural fibroin protein composite film.

5 . Production of Stretched Composite Film and Tension Deformation Test Using a composite film in which polyAla (L-polyAla or T-polyAla) is mixed with the above silk (natural fibroin protein or modified fibroin protein), by the following method. A tension deformation test was performed on a stretched composite film produced by loading a stretch.

Three types of silk, T-polyAla-blended silk, and L-polyAla-blended silk are cut into small pieces (3 mm x 15 mm), immersed in methanol for 5 minutes, and slowly manually uniaxially stretched (IMC-1A11 type, Imoto). It was stretched to 1.25, 1.5, 1.75 or 2 times the length using Mfg. Co., Ltd. (each stretch ratio is 25%, 50%, 75% or 100%). It was fixed on each stretched glass Petri dish with double-sided tape and vacuum dried in a desiccator at room temperature for 3 hours. Using each stretched film, mechanically measured by a tensile tester (small tabletop tester EZ Test series EZ-LX HS, Shimadzu Seisakusho) at a tensile speed of 0.5 mm / min, 25 ° C, and a relative humidity of 55-60%. , Maximum tension, strain (marginal draw ratio) and toughness were calculated from the stress-strain curve. Each measurement was performed in 5 cases, and the mean value and standard deviation were calculated.

6. Wide-angle X-ray diffraction (WAXD) measurement
Wide-angle X-ray diffraction (WAXD) of 100 % stretched silk-only film and film containing polyAla was performed on a BL45XU beamline (SPring-8, Harima).

7. Results
(1) Tension deformation test results for the composite film of natural silk moth fibroin protein 5 wt% or 10 wt% of liner-type polyalanine (L-polyAla) or telechelic-type polyalanine (T-polyAla) was added to the natural silk moth fibroin protein. The results of unstretched physical properties of the film produced by blending 1 wt% or 2.5 wt% are shown in Tables 6, 7 and 5, respectively.

When 5 wt% of liner-type polyalanine (L-polyAla) was added to the natural silk moth fibroin protein, improvement in tensile strength, strain and toughness was observed.

(2) Comparison of physical properties of composite film using natural silk moth (Bombyx mori) silk fibroin protein and modified spider silk fibroin protein L-polyAla 5 wt% or T-polyAla 1wt for silk moth silk fibroin (Bombyx. Mori fibroin) Tension deformation measurements were performed on the composite film containing% (Tables 3 and 5) and the composite film containing L-polyAla 10 wt% or T-polyAla 1 wt% in the modified spider silk protein (Tables 4 and 6). The physical properties were compared.

When L-polyAla 5 wt% was added to silk moth silk fibroin, the strain of the composite film increased by 11.1% and the toughness increased by 16.9%. On the other hand, when 1 wt% of T-polyAla was added to the modified spider silk fibroin protein (ADF Kai-noNR), strain increased by 66.8% and toughness increased by 133%, showing a remarkable improvement in physical characteristics.

(3 ) Tension deformation test results for stretched composite film In the manufacturing process of composite film manufactured by blending 5% of telechelic polyalanine (T-polyAla), the stretching ratio is 0, 25, 50, 75 or 100. FIG. 6 shows the results of a tension deformation test on a composite film produced by further loading% stretching . By performing the stretching operation, the tensile strength, strain and toughness were more clearly increased as compared with the composite film to which the telechelic polyalanine was not added (Fig. 6).

(4) Wide-angle X-ray diffraction (WAXD) measurement results
WAXD results of a stretched composite film produced by stretching a composite film containing L-polyAla or T-polyAla at a ratio of 0, 1, 2.5, 5 or 10 wt% at a stretch ratio of 100%. Each is shown in FIG. FIG. 7a shows the change in WAXD when L-poyAla is added, and FIG. 7b shows the change in WAXD when T-polyAla is added.

The silk-only film shows peaks derived from the (020), (210), (211) planes of the crystal lattice based on the β sheet of ADF3. The interplanar spacing d is 0.51, 0.45, and 0.37 nm, respectively.
T- and L-polyA also show peaks based on β-sheet crystals, but the interplanar spacing d of the (210) plane is 0.43 nm, which is different from that of silk.

FIG. 7a was measured by changing the amount of L-polyAla added as a comparison between 100% stretched films. As the amount added increased, the beak strength of L-polyA based on all β sheets increased. When the addition amount exceeded 5%, the peak intensity of the (210) plane of silk was further increased instead of the peak of the (210) plane of polyalanine. This result suggests that L-polyAla affects the crystal structure of silk due to the structural similarity of the arrangement of L-polyA and silk. It is considered that the promotion of silk crystal growth by L-polyAla leads to the improvement of the stress of the film.

In FIG. 7b, which shows the change in WAXD when T-polyAla is added, the intensity of all peaks of polyalanine increases as the amount of addition is increased, but the intensity of the peak based on the (210) plane of silk is increased. No significant changes were observed. Due to its telechelic structure, T-polyAla is thought to have little effect on silk beta sheets. Since polyalanine alone forms crystals and is dispersed, it is considered that the toughness of the film is improved with a small amount of addition.

In the above results, the WAXD of the composite film in which 5 wt% T-polyAla was blended with the modified spider silk protein increased the peak intensity showing crystals as the blending ratio of T-polyAla or L-polyAla increased. (Figs. 7a and 7a, b). It is considered that T-polyAla or L-polyAla promotes the increase of β-sheet crystals in silk, which affects the physical properties.

Production of composite fiber composition and evaluation of its physical properties 1. Experimental Materials and Methods Reagents and solvents other than the reagents described below were purchased from Wako Pure Chemical Industries, Ltd. (Osaka) and used. The protein powder (SEQ ID NO: 17) was obtained by the following method. Prior to preparing the dope, the protein powder was dried under vacuum at 100 ° C. for 2 hours. Formic acid was used as the solvent for the dope solution. Spinning was performed with a type 1 tabletop spinning device (Spiber Inc., Yamagata) according to the manual of the device. The diameter, tensile stress, tensile strain (strain), and toughness of the spun fiber were confirmed. The diameter was measured with ECLIPSE LV100ND manufactured by Nikon Corporation (Tokyo). Stress and strain were measured and evaluated at INSTRON (Tokyo). Toughness was calculated using Bluehill Software (INSTRON, Tokyo).

2. 2. Production of spider silk protein (PRT799) (synthesis of gene encoding spider silk protein and construction of expression vector)
Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes, a spider silk protein having the amino acid sequence shown in SEQ ID NO: 17 (hereinafter, "" Also called "PRT799") was designed.

The amino acid sequence shown by SEQ ID NO: 17 is a region of 20 domain sequences existing in the amino acid sequence shown by SEQ ID NO: 15 (however, several amino acid residues on the C-terminal side of the region are substituted). The amino acid sequence shown by SEQ ID NO: 21 (including the His tag) is added to the N-terminal to the amino acid sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above four times.

Nucleic acid encoding the designed PRT799 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector.

Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added so that 100 mL of the seed culture medium containing ampicillin (Table 7) had an OD600 of 0.005. The culture solution temperature was kept at 30 ° C., and flask culture was carried out for about 15 hours until the OD600 reached 5, to obtain a seed culture solution.

The seed culture solution was added to the jar fermenter to which 500 ml of the production medium (Table 8 below) was added so that the OD600 was 0.05. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. The culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 1 mM to induce the expression of PRT799. Twenty hours after the addition of IPTG, the culture broth was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the expression of PRT799 was confirmed by the appearance of a band having a size corresponding to PRT799 depending on the addition of IPTG.

(Purification of spider silk protein)
The cells recovered 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL at 60 ° C. Stir with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregate protein (PRT799) obtained after dialysis was recovered by centrifugation. Moisture was removed from the recovered aggregated protein with a lyophilizer to obtain a lyophilized powder of PRT799.

The degree of purification of PRT799 in the obtained freeze-dried powder was confirmed by image analysis of the results of polyacrylamide gel electrophoresis of the powder using Totallab (nonliner dynamics ltd.). As a result, the degree of purification of PRT799 was about 85%.

The modified spider silk protein (PRT799, SEQ ID NO: 17) produced by the above method was used in the following experiment.

3. 3. Experimental method (1) Preparation of dope solution (without L-polyAla and T-polyAla)
The above protein powder was used. Exactly 3.6 g of dry powder was weighed and collected in clear vials. 11.1 g of formic acid was added to the vial and stirred at 40 ° C. overnight to give a clear, deep yellow liquid. The protein concentration in this dope is 24 wt%.

(2) Preparation of dope solution to which L-polyAla or T-polyAla is added
Take 0.036 g (1 wt% of the above protein powder) of polyAla (L-polyAla or T-polyAla) in a vial, add formic acid (11.364 g), and dissolve by stirring at 40 ° C for 1-2 hours. did. After obtaining a clear liquid, protein powder (3.6 g) was added. Stir vigorously at 40 ° C. (usually 8-12 hours) until the protein powder was completely dissolved. The protein concentration in this dope is 24 wt%.

(3) Spinning method Spinning was performed using the above tabletop spinning device (see FIG. 4). The conditions are shown in Tables 9 and 10. Bath 1 was filled with 100% ethanol, baths 2 and 3 were filled with 100% methanol, bath 4 was filled with water (tap water), and buses 5 and 6 were spun in an empty state. The spun fibers were stretched only in bath 4.

(4) Evaluation of physical properties of composite fiber composition The ratio of the tensile speed to the injection speed in the bath draft is 0.6 or 0.5, and the tensile speed in the washing water bath is 7.0 times or 7.8 times at each tensile speed ratio. Tables 11 and 12 show the standard deviations of fiber diameter, tensile strength, strain, toughness, tensile stress and tensile strain (strain) in the case of spinning, respectively.

The smaller the tensile rate ratio (fiber tensile rate / dope solution injection rate) in the bath draft, the longer the agglomeration time and the stronger the agglomeration of the fibers. The above results show that the composite fiber containing 1% of L-polyAla or T-polyAla, respectively, at both the tensile speed ratios of 0.6 and 0.5, improved the tensile strength, strain and toughness. Further, when the tensile speed in the washing water was 7.8 times, the fibers were broken during spinning in the control group to which polyAla was not added in both Tables 11 and 12. These results indicate that the addition of L-polyAla or T-polyAla to a fibroin-derived protein improves the physical properties of the composite fiber.

<Summary>
The results observed above are for either a natural fibroin protein or an artificially modified and produced fibroin-derived protein, a polypeptide or poly such as L-poyAla or T-polyAla with a β-sheet structure. It shows that physical properties such as tensile tension, maximum tensile limit rate and toughness can be improved by blending amino acids to prepare a composite molding composition. Further, the improvement of the physical characteristics of this composite molding composition indicates that the type of polypeptide or polyamino acid having a β-sheet structure differs depending on the type of fibroin-derived protein used in order to bring about the optimum characteristics. there were. Furthermore, it was shown that the blending amounts that bring about the optimum properties differ for each physical property such as tensile tension, maximum tensile limit rate, and toughness.

This result shows that a composite molding composition having improved physical properties can be obtained by changing the raw materials used and the blending amount according to the desired properties of the composite molding composition. ..

Further, in addition to the polypeptide composed of natural amino acids or polyamino acids, a polymer having a β-sheet structure other than those derived from these natural amino acids is blended with a fibroin-derived protein to form the composite molding composition of the present invention. It may be used for manufacturing.

Claims (38)

A composition comprising a fibroin-derived protein and a polyamino acid having a β-sheet structure, wherein the polyamino acid comprises a copolymer of one or two to five amino acids. The composition according to claim 1, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The composition according to claim 1 or 2, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. The invention according to any one of claims 1 to 3, wherein the composition is a composite molding composition, wherein a polyamino acid having a β-sheet structure is blended with a fibroin-derived protein and molded. Composition. The composite molding composition according to claim 4, wherein the composite molding composition retains a β-sheet structure derived from the polyamino acid. The composite molding composition according to claim 4 or 5, wherein the composite molding composition is selected from a composite fiber, a composite film, a composite gel, or a composite molded body. A composite molding composition in which a polyamino acid having a β-sheet structure is blended with a fibroin-derived protein.
The polyamino acid is composed of a copolymer of 1 type or 2 to 5 types of amino acids.
The composite molding composition is selected from composite fibers, composite films, and composite gels.
(i) Steps to prepare a dope solution in which a fibroin-derived protein and a polyamino acid having a β-sheet structure are dissolved.
(ii) A step of molding a composite molding composition from the dope solution,
A composite molded composition, characterized in that it is manufactured by a manufacturing method comprising. The composite molding composition according to claim 7, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The composite molding composition according to claim 7 or 8, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. A stretched composite molding composition, wherein the stretched composite molding composition is selected from composite fibers or composite films.
In addition to steps (i) and (ii) according to claim 7, further
(iii) The step of stretching the composite molding composition obtained in (ii) above in a solvent and drying it.
A stretched composite molded composition, which is produced by a production method comprising. A composite molded composition, wherein the composite molded composition is a composite molded body.
(i) A step of mixing a fibroin-derived protein with a polyamino acid having a β-sheet structure to prepare a mixture.
(ii) The step of applying pressure to the mixture and heating it,
It is a molded body manufactured by a manufacturing method including
A composite molding composition, wherein the polyamino acid comprises a copolymer of one or two to five amino acids. The composite molding composition according to claim 11, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The composite molding composition according to claim 11 or 12, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. The fibroin-derived protein
(i) Natural fibroin-derived protein and / or
(ii) The composite molding composition according to any one of claims 4 to 13, which is selected from modified fibroin-derived proteins. The natural fibroin-derived protein is selected from the group consisting of silk fibroin-derived silk protein (silk fibroin protein), spider silk fibroin-derived spider silk protein (spider silk fibroin protein), and hornet silk fibroin-derived hornet silk protein. The composite molding composition according to claim 14, wherein the silk composition is at least one kind. The composite molding composition according to claim 14, wherein the modified fibroin-derived protein is a modified fibroin-derived protein containing a domain sequence represented by the following formula (I).

However, in the domain sequence, at least one or more glycine residues in REP are replaced with different amino acid residues as compared with naturally occurring fibroin, and the content of glycine residues is reduced. Modified fibroin protein with sequence:
[In Formula I,
(A) _{The n} motif indicates an amino acid sequence composed of 2 to 20 amino acid residues, and
(A) The number of alanine residues is 40% or more of the total number of amino acid residues in the _n motif.
REP shows an amino acid sequence consisting of 2 to 200 amino acid residues.
m is an integer from 2 to 300
Multiple (A) _n motifs may have the same amino acid sequence or different amino acid sequences.
A plurality of REPs may have the same amino acid sequence or different amino acid sequences.] In the modified fibroin-derived protein of claim 16, the domain sequence is GGX and GPGXX in REP as compared to naturally occurring fibroin (where X represents an amino acid residue other than glycine). In at least one motif sequence selected from, the glycine residue has an amino acid sequence corresponding to the substitution of one glycine residue in at least one or more of the motif sequences with another amino acid residue, and the glycine residue is A composite molding composition, wherein the proportion of the motif sequence substituted with another amino acid residue is 10% or more with respect to the total motif sequence. In the modified fibroin-derived protein according to claim 16 or 17, the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units from the N-terminal side to the C-terminal side is determined. When the number of amino acid residues of a REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other _REP is 2 to 3.5. When the total value of the sum of the number of amino acid residues of the motif-REP] unit is x and the total number of amino acid residues of the domain sequence is y, the maximum value of x / y (%) is 20% or more. A composite molding composition, characterized in that it is present. The modified fibroin-derived protein represented by the formula (I) is a modified fibroin-derived protein having an amino acid sequence represented by SEQ ID NOs: 14 to 19. The composite molding composition according to any one item. The composite molding composition is characterized in that the fibroin-derived protein is a fibroin-derived protein having 80% or more homology with the amino acid sequence of the fibroin-derived protein according to any one of claims 14 to 19. thing. The composite molding composition according to any one of claims 4 to 20, wherein the polyamino acid having a β-sheet structure is polyalanine. The composite molding composition according to claim 21, wherein the polyalanine is selected from liner-type polyalanine or telechelic-type polyalanine. A method for producing a composite molding composition selected from composite fibers, composite films, and composite gels, in which a polyamino acid having a β-sheet structure is blended with a fibroin-derived protein.
(i) Preparation of a dope solution in which a fibroin-derived protein and a polyamino acid having a β-sheet structure are dissolved,
(ii) A step of producing the composite molding composition by spinning or stretching the dope solution.
Including
A production method, wherein the polyamino acid is composed of a copolymer of 1 type or 2 to 5 types of amino acids. The production method according to claim 23, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The production method according to claim 23 or 24, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. A method for producing a composite molded product in which a polyamino acid having a β-sheet structure is blended with a fibroin-derived protein.
(i) A step of mixing a powdery fibroin-derived protein with a polyamino acid having a powdery β-sheet structure to prepare a mixture.
(ii) The step of applying pressure to the mixture and heating it,
Including
A production method, wherein the polyamino acid is composed of a copolymer of 1 type or 2 to 5 types of amino acids. The production method according to claim 23, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The production method according to claim 26 or 27, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. A method for producing a stretched composite molded composition.
(i) A step of stretching the composite molding composition produced by the production method according to any one of claims 23 to 28 in a solvent, and
(ii) A step of drying the stretched composite molded composition,
A manufacturing method comprising: The fibroin-derived protein
(i) Natural fibroin-derived protein and / or
(ii) The production method according to any one of claims 23 to 29, which is selected from modified fibroin-derived proteins. The natural fibroin-derived protein is selected from the group consisting of silk fibroin-derived silk protein (silk fibroin protein), spider silk fibroin-derived spider silk protein (spider silk fibroin protein), and hornet silk fibroin-derived hornet silk protein. 30. The production method according to claim 30, wherein the silk is at least one kind. The production method according to any one of claims 23 to 31, wherein the polyamino acid having a β-sheet structure is polyalanine. 32. The production method according to claim 32, wherein the polyalanine is selected from liner-type polyalanine or telechelic-type polyalanine. A fibroin-derived protein is mixed with a polyamino acid having a β-sheet structure.
A method for improving the physical properties of a composite molding composition, wherein the polyamino acid comprises a copolymer of one or two to five amino acids. 34. The method of claim 34, wherein the degree of polymerization of the polyamino acid is in the range of 5 to 50. The method according to claim 34 or 35, wherein the compounding ratio of the polyamino acid is in the range of 1 to 10.0%. A method for improving the physical properties of a composite molding composition, which comprises the method according to any one of claims 34 to 36, further comprising stretching the composite molding composition in a solvent and then drying it. .. The method according to any one of claims 34 to 37, wherein the improvement in physical properties is an increase in tensile strength, an increase in strain and / or an increase in toughness.

Images